The tetracyclines are broad spectrum antimicrobial agents that are widely used in human and veterinary medicine (Schappinger et al., “Tetracyclines: Antibiotic Action, Uptake, and Resistance Mechanisms” Arch. Microbiol. 165:359-69, 1996; Mitscher, Medicinal Research Series, Vol. 9, The Chemistry of the Tetracycline Antibiotics, Marcel Dekker Inc. New York, 1978). The total production of tetracyclines by fermentation or semi-synthesis is measured in the thousands of metric tons per year. The first tetracycline, chlorotetracycline (1) (AUREOMYCIN™), was isolated from the soil bacterium Streptomyces aureofaciens by Lederle Laboratories (Wyeth-Ayerst Research) in 1945 (Duggar, Ann. N.Y. Acad. Sci. 51:177-181, 1948; Duggar, Aureomycin and Preparation of Some, U.S. Pat. No. 2,482,055, 1949; incorporated herein by reference). Oxytetracycline (2) was isolated soon after from S. rimosus by scientists at Pfizer Laboratories (Finlay et al., Science 111:85, 1950). The structures of chlorotetracycline and oxytetracycline were elucidated by scientists at Pfizer in collaboration with R. B. Woodward and co-workers at Harvard University (Hochstein et al., J. Am. Chem. Soc. 74:3708-3709, 1952; Hochstein et al., J. Am. Chem. Soc. 75:5455-75, 1953; Stephens et al., J. Am. Chem. Soc. 74:4976-77, 1952; Stephens et al., J. Am. Chem. Soc. 76:3568-75, 1954). Tetracycline (3) was later prepared by the hydrogenolysis of chlorotetracycline and was found to retain the antimicrobial activity of chlorotetracycline and oxytetracycline and had increased stability (Boothe et al. J. Am. Chem. Soc. 75:4621, 1953; Conover et al. J. Am. Chem. Soc. 75:4622-23, 1953). Tetracycline was later found to be a natural product of S. aureofaciens, S. viridofaciens, and S. rimosus.

The primary tetracyclines of clinical importance today include tetracycline (3) (Boothe et al., J. Am. Chem. Soc. 75:4621, 1953), oxytetracycline (2, TERRAMYCIN™) (Finlay et al., Science 111:85, 1950), (−)-doxycycline (Stephens et al., J. Am. Chem. Soc. 85:2643, 1963), (−)-minocycline (Martell et al., J. Med. Chem. 10:44, 1967; Martell et al., J. Med. Chem. 10:359, 1967), and tigecycline. The tetracyclines exert their antimicrobial activity by inhibition of bacterial protein synthesis (Bentley and O'Hanlon, Eds., Anti-Infectives: Recent Advances in Chemistry and Structure-Activity Relationships The Royal Society of Chemistry: Cambridge, UK, 1997). Most tetracyclines are bacteriostatic rather than bactericidal (Rasmussen et al., Antimicrob. Agents Chemother. 35:2306-11, 1991; Primrose and Wardlaw, Ed. “The Bacteriostatic and Bacteriocidal Action of Antibiotics” Sourcebook of Experiments for the Teaching of Microbiology Society for General Microbiology, Academic Press Ltd., London, 1982). It has been proposed that after tetracycline passes through the cytoplasmic membrane of a bacterium it chelates Mg+2, and this tetracycline-Mg+2 complex binds the 30S subunit of the bacterial ribosome (Goldman et al., Biochemistry 22:359-368, 1983). Binding of the complex to the ribosome inhibits the binding of aminoacyl-tRNAs, resulting in inhibition of protein synthesis (Wissmann et al., Forum Mikrobiol. 292-99, 1998; Epe et al., EMBO J. 3:121-26, 1984). Tetracyclines have also been found to bind to the 40S subunit of eukaryotic ribosome; however, they do not achieve sufficient concentrations in eukaryotic cells to affect protein synthesis because they are not actively transported in eukaryotic cells (Epe et al., FEBS Lett. 213:443-47, 1987).
Structure-activity relationships for the tetracycline antibiotics have been determined empirically from 50 years of semi-synthetic modification of the parent structure (Sum et al., Curr. Pharm. Design 4:119-32, 1998). Permutations of the upper left-hand portion of the natural product, also known as the hydrophobic domain, have provided new therapeutically active agents, while modifications of the polar hydrophobic domain result in a loss of activity. However, semi-synthesis by its very nature has limited the number of tetracycline analogs that can be prepared and studied.

The tetracyclines are composed of four linearly fused six-membered rings with a high density of polar functionality and stereochemical complexity. Previous approaches to the synthesis of tetracyclines typically proceeded via a stepwise assembly of the tetracyclic ring system. In 1962, Woodward and co-workers reported the first total synthesis of racemic 6-desmethyl-6-deoxytetracycline (sancycline, 4), the simplest biologically active tetracycline (Conover et al., J. Am. Chem. Soc. 84:3222-24, 1962). The synthetic route was a remarkable achievement for the time and proceeded by the stepwise construction of the rings in a linear sequence of 22 steps (overall yield˜0.003%). The first enantioselective synthesis of (−)-tetracycline (3) from the A-ring precursor D-glucosamine (34 steps, 0.002% overall yield) was reported by Tatsuda and co-workers in 2000 (Tatsuta et al., Chem. Lett. 646-47, 2000). Other approaches to the synthesis of tetracycline antibiotics, which have also proceeded by the stepwise assembly of the ABCD ring system beginning with D or CD precursors, include the Shemyakin synthesis of (±)-12a-deoxy-5a,6-anhydrotetracycline (Gurevich et al., Tetrahedron Lett. 8:131, 1967; incorporated herein by reference) and the Muxfeldt synthesis of (±)-5-oxytetracycline (terramycin, 22 steps, 0.06% yield) (Muxfeldt et al., J. Am. Chem. Soc. 101:689, 1979; incorporated herein by reference). Due to the length and poor efficiency of the few existing routes to tetracyclines, which were never designed for synthetic variability, synthesis of tetracycline analogs is still limited.

More recently, a novel convergent synthetic route to tetracyclines and various analogs, including pentacycline and heterocycle-containing tetracyclines, has been developed by Myers and co-workers. See US 2005/0282787, published Dec. 22, 2005; incorporated herein by reference; and Charest et al., Science, 308:395-398, 15 Apr. 2005; Charest et al., J. Am. Chem. Soc. 127:8292-93, 2005. This route proceeds through the highly functionalized chiral enone intermediate (5) which is prepared starting from benzoic acid in ten steps (11% yield, >95% ee) (Charest et al., Science 308:395-398, Apr. 15, 2005; Charest et al., J. Am. Chem. Soc. 127:8292-8293, 2005; Myers et al., Org. Lett. 3(18):2923-26, 2001). A second generation route to the enone intermediate (5) was later developed starting from an isoxazole aldehyde. The second generation route yields the enone in eight steps in an improved yield. See US 2009/0093640, published Apr. 9, 2009; U.S. 60/850,859, filed Oct. 11, 2006; and WO2008/127361, published Oct. 23, 2008; each of which is incorporated herein by reference.
Several approaches were developed to react the enone 5 with a toluate (6), benzylic halide, or benzocyclobutenol (8) to form the tetracycline core ring system. The first approach involves the reaction of the enone with an anion formed by the deprotonation of a toluate (6) or metallation of a benzylic halide as shown below.
A second approach involves reacting the enone in a Diels-Alder-type reaction with a diene (7) or a benzocyclobutenol (8).
In these approaches, the chiral enone provides the functionalized A and B rings of the tetracycline core, and the D-ring is derived from the toluate (6), benzylic halide, or benzocyclobutenol (8). In bringing the two portions of the tetracycline core together the C-ring is formed, preferably in a stereoselective manner. These new synthetic approaches to tetracycline analogs not only allow for the stereoselective and efficient synthesis of a wide variety of tetracycline analogs never before prepared, but they also allow for preparation of tetracycline analogs in which the D-ring is replaced with a heterocycle, 5-membered ring, or other ring systems. The new methodologies also allow for the preparation of various pentacyclines or higher cyclines containing aromatic and non-aromatic carbocycles and heterocycles. See U.S. patent application, US2005/0282782, published Dec. 1, 2005; PCT Application WO 05/112985, published Dec. 1, 2005; and U.S. Provisional Patent Application Ser. No. 60/790,413, filed Apr. 7, 2006; each of which is incorporated herein by reference.
A second generation route to the functionalized chiral enone intermediate (9) useful in synthesizing tetracyclines was also recently described by Myers and coworkers (US 2009/0093640, published Apr. 9, 2009; U.S. 60/850,859, filed Oct. 11, 2006; and WO2008/127361, published Oct. 23, 2008; each of which is incorporated herein by reference).
The route yields the highly functionalized chiral enone intermediate (9) in eight steps from an isoxazole aldehyde and allows for the preparation of tetracycline analogs in higher yields that previously attainable.
Although the above approaches to tetracycline analogs are much more efficient than earlier approaches and allow for synthetic variability, there remains a need for improving the efficiency and versatility of these routes to new tetracycline analogs.